PROJECT SUMMARY HIV-Associated Neurocognitive Disorder (HAND) persists in up to 50% of the HIV-positive population, often in spite of low or undetectable viral loads due to effective antiretroviral therapy (ART). Microglia are the resident macrophages in the central nervous system (CNS) and are thought to be a persistent reservoir of HIV in ART treated patients. Microglia secrete a panoply of factors that impact neuronal function and these secreted factors are altered during HIV infection of microglia. In addition to HIV virus-host interactions mediated by microglia, many exogenous factors can modify these interactions or directly impact the CNS itself, making it difficult to dissect the underlying causes of neural pathology leading to HAND. To establish a physiologically- relevant platform to investigate the dynamics of HIV infection in the context of ART and other co-factors, we will use human induced pluripotent stem cells to generate human microglia that we can co-culture with our well-characterized 3D model of the developing cerebral cortex. This integrated 3D cerebral organoid model will recapitulate many of the critical cell populations thought to contribute to HAND-related pathology including glutamatergic neurons, astrocytes, and microglia. We will evaluate the efficacy of the cerebral organoids to model HIV infections by measuring HIV replication kinetics and suppression, as well as HIV protein expression in different cell types (Aim 1). We will then measure the impact of HIV on neuronal development and function at several time points following acute exposure (Aim 2). We will perform morphological and electrophysiological analyses and RNA sequencing at both the single cell and population level to determine the cell type-specific transcriptional responses to HIV exposure. Finally, we will evaluate the impact of exogenous drugs on these measures of viral replication and neuronal function (Aim 3). As a proof-of-concept, we will test a preferred regimen of ART, as well as cannabinoids, both with and without HIV exposure to begin to dissect the impact of these common co-factors on cellular properties that may contribute to pathology underlying HAND. Successful completion of these experiments will result in a validated platform to model the dynamics of neuroinflammatory disorders using brain region-specific cerebral organoids integrated with human microglia.